As part of a broad program to study aerobic energy metabolism in parasites and mammals, current interest is focused on the role of iron-sulfur proteins in the respiratory chain of Entamoeba histolytica. Chemical analyses of a partially purified amebal diaphorase (which catalyzes the oxidation of NAD(P)H with molecular oxygen as final electron acceptor) disclosed the presence of non-heme iron and acid-labile sulfide in a molar ratio of 1:1. When examined by low-temperature electron parametic resonance (EPR) spectroscopy, the diaphorase exhibited an unusually symmetrical, narrow signal at g equals 2.036. The EPR spectra of diaphorase preparations isolated from amebae grown with 57Fe showed a broadened signal, and analyses by mass spectroscopy confirmed that isotopic substitution had occurred, thus providing unequivocal evidence that the EPR signal arises from an iron-sulfur center in the amebal diaphorase. Other studies demonstrated that non-heme iron also functions in the bacterial respiratory chain. Mammalian studies included a collaborative investigation of the interrelationship of heme synthesis and mitochondrial bioenergetics. Future work will attempt to further purify, characterize and elucidate the role of iron-sulfur proteins in electron and energy transfer reactions.